The Notch pathway is a conserved signaling mechanism that functions to modulate cell- fate decisions. The overall objectives of this proposal are to define roles for Notch in cell- fate determination during lymphatic remodeling, and tumor lymphangiogenesis. Our general hypothesis is that Notch activity is necessary to assist in lymphatic remodeling. We hypothesize unique roles for different Notch family members in lymphatic growth and development, with a focus on Notch1, Notch3, and Notch4. We have discovered that VEGFR-3, a lymphatic regulator, is transciptionally regulated by Notch1 and Notch4. In contrast, a complex regulation of Prox-1 by Notch was uncovered with opposing roles for Notch1 and Notch4. Our general strategy will use a combination of in vitro lymphangiogenesis assays and mouse modeling to define the consequences of altering Notch activity in lymphatic endothelial cells. In addition, our preliminary studies implicate Notch as a regulator of tumor lymphatic growth and a factor controlling VEGFR-3 expression in tumor vessels and lymphatics. In Aim I, we evaluate Notch function in isolated lymphatic endothelial cells with the goal of determining if Notch regulation of VEGFR-3 and Prox1 is key to lymphatic endothelial cell behavior. In Aim II, we will analyze Notch mutant mice and activate Notch in dermal lymphatic endothelial cells to determine if Notch promotes lymphangiogenesis, lymphatic remodeling or lymphatic integrity. In Aim III, we use a Notch inhibitor developed in the lab, a secreted antagonist of ligand-dependent Notch signaling, to evaluate if VEGF-C-mediated tumor lymphangiogenesis requires Notch. Our overall goal is to study both developmental and pathological lymphangiogenesis to better understand Notch function in lymphatics. PUBLIC HEALTH RELEVANCE: Notch signaling is fundamental to proper vascular development. We present data strongly implicating Notch as a regulator of lymphatic development and pathologies. Thus, the studies outline in our proposal will aid us in understanding the functions of Notch in lymphatic differentiation and may be critical to developing strategies and therapeutics to correct lymphatic disorders or block tumor lymphangiogenesis.